The ability to modulate protein activities has long been the hallmark of small molecule drug discovery and development, and the success of this traditional therapeutic approach is unquestioned. However, the number and nature of small molecule drug targets are more limiting than would be ideal and have less target specificity and more off-target side effects that will likely make for significant commercial and regulatory challenges in the years ahead. A newer technology for inhibiting protein activity that has received acceptance is siRNA-mediated gene silencing. The mechanism for siRNA inhibition is post-transcriptional and pre-translational. It has the advantage of being relatively selective for target RNA sequences but, like small molecules, suffers from off-target side effects.
Kinases are enzymes that catalyze the addition of phosphate to a molecule. The addition of phosphate by a kinase is called phosphorylation. When the kinase substrate is a protein molecule, the amino acids commonly phosphorylated are serine, threonine and tyrosine. Phosphatases are enzymes that remove phosphate from a molecule. The removal of phosphate is called dephosphorylation. Kinases and phosphatases often represent competing forces within a cell to transmit, attenuate, or otherwise modulate cellular signals and cellular control mechanisms. Kinases and phosphatases have both overlapping and unique natural substrates. Cellular signals and control mechanisms, as regulated by kinases, phosphatases, and their natural substrates are a target of research tool design and drug design.
Mammalian mitogen-activated protein kinase (MAPK) and extracellular-signal-regulated kinase (ERK) are the same enzyme, herein referred to as ERK. ERK has two isoforms, both of which can phosphorylate serine and threonine residues in protein or peptide substrates. Use of the term ERK herein encompasses both ERK isoforms. Many cellular substrates of ERK have been identified. Furthermore, polypeptides have been used to examine ERK substrate specificity. While polypeptides and variants thereof have been studied as individual substrates or ligands, mixed ligands linked together as polyligands that modulate ERK activity have not been demonstrated before this invention. An aspect of the invention is to provide novel, modular, inhibitors of ERK activity by modifying one or more natural substrates by truncation and/or by amino acid substitution. A further aspect of the invention is the subcellular localization of an ERK inhibitor, ligand, or polyligand by linking to a subcellular localization signal. Examples of ERK substrates and/or regulators include those described in the following references: Adams, et al. 2000 J Neurochem 75:2277-87, Arnaud, et al. 2004 J Immunol 173:3962-71, Chung, et al. 1997 Mol Cell Biol 17:6508-16, Clark-Lewis, et al. 1991 J Biol Chem 266:15180-4, Eymin, et al. 2006 Cell Cycle 5:759-65, Fantz, et al. 2001 J Biol Chem 276:27256-65, Garcia, et al. 2002 Embo J 21:5151-63, Gille, et al. 1995 Embo J 14:951-62, Haycock, et al. 1992 Proc Natl Acad Sci USA 89:2365-9, Hedges, et al. 2000 Am J Physiol Cell Physiol 278:C718-26, Hindley, et al. 2002 J Cell Sci 115:1575-81, Howell, et al. 1991 Mol Cell Biol 11:568-72, Ishibe, et al. 2004 Mol Cell 16:257-67, Jacobs, et al. 1999 Genes Dev 13:163-75, Jacque, et al. 1998 Embo J 17:2607-18, Kelemen, et al. 2002 J Biol Chem 277:8741-8, Kolch 2000 Biochem J 351 Pt 2:289-305, Lefebvre, et al. 2002 J Cell Biol 157:603-13, Lin, et al. 1999 J Biol Chem 274:15971-4, Matallanas, et al. 2006 Mol Cell Biol 26:100-16, Matsuura, et al. 2005 Biochemistry 44:12546-53, Matter, et al. 2002 Nature 420:691-5, Missero, et al. 2000 Mol Cell Biol 20:2783-93, Morton, et al. 2004 FEBS Lett 572:177-83, Pandey, et al. 2005 Mol Cell Biol 25:10695-710, Sanghera, et al. 1990 FEBS Lett 273:223-6, Schaeffer, et al. 1999 Mol Cell Biol 19:2435-44, Songyang, et al. 1996 Mol Cell Biol 16:6486-93, Soond, et al. 2005 J Cell Sci 118:2371-80, Terret, et al. 2003 Development 130:5169-77, Veeranna, et al. 1998 J Neurosci 18:4008-21, Xu, et al. 2001 Mol Cell Biol 21:2981-90, Zhang, et al. 2001 J Biol Chem 276:14572-80, and MAP Kinase Substrate Peptide Catalog #2-125 Lot #23369 (Upstate, Lake Placid, N.Y.).
Design and synthesis of polypeptide ligands that modulate calcium/calmodulin-dependent protein kinase and that localize to the cardiac sarco(endo)plasmic reticulum was performed by Ji et al. (J Biol Chem (2003) 278:25063-71). Ji et al. accomplished this by generating expression constructs that localized calcium/calmodulin-dependent protein kinase inhibitory polypeptide ligands to the sarcoplasmic reticulum by fusing a sarcoplasmic reticulum localization signal derived from phospholamban to a polypeptide ligand. See also U.S. Pat. No. 7,071,295.